Structural Basis for Thermophilic Protein Stability: Structures of Thermophilic and Mesophilic Malate Dehydrogenases

Dalhus, Bjørn; Saarinen, Markuu; Sauer, Uwe H.; Eklund, Pär; Johansson, Kenth; Karlsson, Andreas; Ramaswamy, S.; Bjørk, Alexandra; Synstad, Bjørnar; Naterstad, Kristine; Sirevåg, Reidun; Eklund, Hans

doi:10.1016/s0022-2836(02)00050-5

Cited by 102 publications

(110 citation statements)

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“…For the operation of this cycle, both NADH and NADPH are required: malate dehydrogenase preferentially uses NADH (Dalhus et al 2002) and isocitrate dehydrogenase NADPH (Lebedeva et al 2002). For autotrophic growth, the reaction center complex reduces Fd and electrons are channeled to NAD(P) + via FNR.…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of ferredoxin-NADP+ reductase encoded by Bacillus subtilis yumC

2004

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From Bacillus subtilis cell extracts, ferredoxin-NADP + reductase (FNR) was purified to homogeneity and found to be the yumC gene product by N-terminal amino acid sequencing. YumC is a ~94 kDa homodimeric protein with one molecule of non-covalently bound FAD per subunit. In a diaphorase assay with 2,6-dichlorophenol-indophenol as an electron acceptor, the affinity to NADPH was much higher than to NADH, with K m values of 0.57 vs. >200 µM. K cat values of YumC with NADPH were 22.7 and 35.4 s -1 in diaphorase and in a ferredoxin-dependent NADPH-cytochrome c reduction assay, respectively. The cell extracts contained another diaphorase-active enzyme, the yfkO gene product, but its affinity for ferredoxin was very low. The deduced YumC amino acid sequence has high identity to that of the recently identified Chlorobium tepidum FNR. A genomic database search indicated that there are more than 20 genes encoding proteins that share a high level of amino acid sequence identity with YumC and annotated variously as NADH oxidase, thioredoxin reductase, thioredoxin reductase-like protein, etc. These genes are found notably in Gram-positive bacteria except for Clostridia, and less frequently in archaea and proteobacteria. We propose that YumC and C. tepidum FNR constitute a new group of FNR which should be added to the already established plant type, bacteria type, and mitochondria type FNR groups.

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Section: Discussionmentioning

confidence: 99%

Purification and characterization of ferredoxin-NADP+ reductase encoded by Bacillus subtilis yumC

2004

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“…It is folded into two domains: the N-terminal domain contains the cofactor binding site and the C-terminal domain contains the active site . In hyperthermophilic MDHs, the hydrophobic residues are mainly positioned in the protein core (Irimia et al, 2004) and an increase in the packing density decreases the protein volume (Dalhus et al, 2002).…”

Section: Crystallization Cofactor Binding Sites and Catalytic Domainsmentioning

confidence: 99%

Function, kinetic properties, crystallization, and regulation of microbial malate dehydrogenase

Takahashi-Íñiguez

Aburto-Rodríguez

Vilchis-González

et al. 2016

J. Zhejiang Univ. Sci. B

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Abstract:Malate dehydrogenase (MDH) is an enzyme widely distributed among living organisms and is a key protein in the central oxidative pathway. It catalyzes the interconversion between malate and oxaloacetate using NAD + or NADP + as a cofactor. Surprisingly, this enzyme has been extensively studied in eukaryotes but there are few reports about this enzyme in prokaryotes. It is necessary to review the relevant information to gain a better understanding of the function of this enzyme. Our review of the data generated from studies in bacteria shows much diversity in their molecular properties, including weight, oligomeric states, cofactor and substrate binding affinities, as well as differences in the direction of the enzymatic reaction. Furthermore, due to the importance of its function, the transcription and activity of this enzyme are rigorously regulated. Crystal structures of MDH from different bacterial sources led to the identification of the regions involved in substrate and cofactor binding and the residues important for the dimer-dimer interface. This structural information allows one to make direct modifications to improve the enzyme catalysis by increasing its activity, cofactor binding capacity, substrate specificity, and thermostability. A comparative analysis of the phylogenetic reconstruction of MDH reveals interesting facts about its evolutionary history, dividing this superfamily of proteins into two principle clades and establishing relationships between MDHs from different cellular compartments from archaea, bacteria, and eukaryotes.

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“…The dependence of a structure's sequence spectrum on its contact trace is a phenomenon that may have measurable biological consequences. Thermophilic organisms living at extremely high temperatures must constantly meet an extraordinary demand for thermally stable biomachinery (15,16) as compared with their mesophilic counterparts who live at more moderate temperatures. We speculated that the force of natural selection may have biased the structural proteomes of thermophilic organisms toward selecting folds of higher contact trace, because these structures are more designable and thus more mutationally plastic and adaptable in a high-temperature environment.…”

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confidence: 99%

“…Previously, studies investigating the cause of thermostability (23) in proteins from thermophilic eubacteria have either focused in detail on the variation of relatively small families of folds from thermophile to mesophile (24,25) or else have directed their attention principally toward particular sequence-based means for thermal stabilization (26,27), such as salt-bridge formation (28,29) or disulfide bridges (15). In contrast to previous studies, here we focus on comparative analysis of patterns of fold usage across whole thermophilic and mesophilic proteomes by calculating the normalized contact trace distributions for sets of fully sequenced thermophilic and mesophilic genomes.…”

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confidence: 99%

Natural selection of more designable folds: A mechanism for thermophilic adaptation

England

Shakhnovich

2003

Proc. Natl. Acad. Sci. U.S.A.

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An open question of great interest in biophysics is whether variations in structure cause protein folds to differ in the number of amino acid sequences that can fold to them stably, i.e., in their designability. Recently, we have shown that a novel quantitative measure of a fold's tertiary topology, called its contact trace, strongly correlates with the fold's designability. Here, we investigate the relationship between a fold's contact trace and its relative frequency of usage in mesophilic vs. thermophilic eubacteria. We observe that thermophilic organisms exhibit a bias toward using folds of higher contact trace when compared with mesophiles. We establish this difference both for the distributions of folds at the whole-proteome level and also through more focused structural comparisons of orthologous proteins. Our findings suggest that thermophilic adaptation in bacterial genomes occurs in part through natural selection of more designable folds, pointing to designability as a key component of protein fitness.U nderstanding the adaptations that enable thermophilic organisms to survive at extreme temperatures is a challenge that has interested researchers since 1897 (1), and great strides have been made along this line of inquiry since the recent publications of complete genomes for several hyperthermophilic species (2). However, most research in this area has focused on amino acid sequence variations that increase the thermodynamic stability of thermophilic proteins while leaving their structures unchanged (2, 3). Far less is known about what structural differences exist between thermophilic and mesophilic proteomes. One interesting possibility is that a thermophilic bias in structure may manifest as a preference for folds that are able to accommodate a large number of low-energy sequences, because such folds have a higher probability of being able to maintain their stability while adapting by mutation to new pressures. This hypothesis relates to another problem of great interest in biophysics: how the structural topology of a protein fold affects its designability (4-9). Here, we present results that point to an important connection between these two problems. Making use of what is known analytically about the relationship between a fold's topology and its designability and thermostability, we report an adaptation mechanism of thermophillic bacteria: one that proceeds by selection of more designable folds.Recently, a new theoretical treatment of designability has been developed within the framework of a residue-residue contact Hamiltonian (10). This Hamiltonian is a well established model for protein energetics that defines the conformational energy of a polypeptide chain as the sum of the pairwise interaction energies of all of the amino acid pairs whose ␣ carbons are separated by a distance less than some contact cutoff, typically chosen to be Ϸ7.5 Å (11). More formally, for a chain of length N with a monomer alphabet containing M amino acid types (of course, M ϭ 20 for real proteins), we can define the ene...

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Structural Basis for Thermophilic Protein Stability: Structures of Thermophilic and Mesophilic Malate Dehydrogenases

Cited by 102 publications

References 59 publications

Purification and characterization of ferredoxin-NADP+ reductase encoded by Bacillus subtilis yumC

Purification and characterization of ferredoxin-NADP+ reductase encoded by Bacillus subtilis yumC

Function, kinetic properties, crystallization, and regulation of microbial malate dehydrogenase

Natural selection of more designable folds: A mechanism for thermophilic adaptation

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